Stem Cells of Skeletal Muscle
The quest for a cell able to restore muscle structure and function in dystrophic patients started in 1961 with the identification of satellite cells (1). Although satellite cells remain the cell type that by and large retain the main myogenic activity in adult muscle (2, 3), some of their biological features limit their potential use for the treatment of primary myopathies. In fact satellite cells lack the ability to cross the muscle endothelium when delivered systemically and must be injected intramuscularly every 2 mm3 of all, or at least of life essential, muscles of the patients, since this is the maximal distance they apparently can migrate from the site of injection (4). This feature alone makes their use in cell therapy protocols extremely difficult, at least with current technology, also considering that the large majority of injected cells are lost within the first day (5). A second problem is represented by the reduced proliferation potency of satellite cells from dystrophic patients and also by the recent observation that in vitro expansion reduces their in vivo differentiation potency (6).
The demonstration that other cell types, resident in the bone marrow or in the vascular niche of other tissues, can differentiate into skeletal muscle in vitro or in vivo created an alternative possibility for the cell therapy of muscular dystrophy (7). The ideal cell population should be i) easily obtainable from accessible anatomical sites, ii) expandable in vitro to the large number of cells required for systemic treatment (109 or more), iii) easily transducible with viral vectors, vi) able to reach skeletal muscle through a systemic route and, finally v) able to differentiate into skeletal muscle cells in vivo while maintaining a self-renewal ability. Of the many types of recently identified and characterized mesoderm stem cells, many show one or more of these features. However, in general their characteristics have not been investigated systematically. By contrast, embryonic mouse mesoangioblasts have been shown to restore muscle morphology and function in a mouse model of muscular dystrophy (8). Human and mouse cells dramatically differ in the ability to extensively proliferate in vitro and it is therefore essential to test whether human cells corresponding to embryonic mouse mesoangioblasts exist in fetal or post-natal human tissues and, if so, whether they show features that may allow to predict a successful use in cell therapy protocols for muscular dystrophy.
In the present study, the cells originating from normal and dystrophic adult human skeletal muscle are named periangioblasts, and can be expanded in vitro for about 20 population doublings before undergoing senescence as diploid non tumorigenic cells; they can be transduced with viral vectors expressing mini dystrophin or other therapeutic genes and then induced to differentiate into skeletal muscle.
When transplanted into dystrophic immune-incompetent mice they give rise to large numbers of new fibers expressing human dystrophin. The cells of the present invention, differ from any other mesoderm stem/progenitor cells because of a) their source (blood vessels), b) their method of isolation (explant rather than proteolytic digestion) and c) their myogenic differentiation potency which is strikingly higher than any other cell in the body, beside resident satellite cells.
Periangioblasts express some of the proteins that leukocytes use to adhere to and cross the endothelium and thus can diffuse into the interstitium of skeletal muscle when delivered intra-arterially. This is a distinct advantage over resident satellite cells that cannot do the same.
Therefore catheter mediated delivery to the succlavia, the diaphragmatic and the iliac arteries should allow periangioblasts from skeletal muscle to reach and colonize muscles that are essential for motility and breathing.
More importantly, when induce to differentiate in vitro, periangioblasts spontaneously differentiate up to 40% of the population, an efficiency far superior to any other non myogenic cell tested so far and second only to resident satellite cells which however cannot be delivered through the circulation. Although not yet tested in a systematic comparative way, the number of dystrophin positive muscle fibers produced in vivo by periangioblasts is far higher than what reported previously by other authors.
Thus, the human cell periangioblast population of the present invention fulfils all the criteria for a successfully cell therapy protocol in muscular disorders such as Duchenne muscular dystrophy. Periangioblasts can be easily isolated from the biopsy that is used for diagnosis. A needle biopsy is a tolerable surgery that can be repeated every few years to further the protocol therapy.
Stem Cells of Cardiac Muscle
The post-infarction ventricular remodeling is characterized by progressive expansion of the initial infarct area and of the left ventricular lumen, with cardiomyocyte replacement by fibrous tissue deposition in the ventricular wall. One approach proposed to reverse myocardial remodeling is regeneration of cardiac myocytes using stem cells (35). Different groups have already reported the isolation of cardiac stem-like cells based on distinct cell surface markers such as Sca-1 or c-Kit (36, 37); these cells are able to restore cardiac function after ischemic injury although with variable efficacy. However their spontaneous cardiac differentiation is low and they also differentiate into other tissue types of the heart (36-39) suggesting that they represent the in vitro expansion of a pluripotent progenitor, that still requires specific signals to undergo terminal cardiac differentiation. On the other hand, Isl-1 expressing progenitors appear to be committed to cardiac differentiation only but still require interactions with other. cells for both proliferation and differentiation (38). The emerging scenario reveals an unforeseen complexity where different types of progenitors may be identified and eventually isolated at different stages of their differentiation process. It is also becoming clear that a significant part of the beneficial effect that most of these cells exert on the infarcted heart is due to the secretion of factors that increase survival of residual myocardium and/or favor angiogenesis (40). This was for example the case of embryonic mesoangioblasts whose transplantation resulted in a 50% recovery of cardiac function but whose differentiation into new cardiomyocytes was rare (41).
In the present invention, adult mouse and human cardiac muscle biopsies were performed allowing, through mechanical and not enzymatic dissociation method, the isolation of cells denominated adult cardiac mesoangioblasts. It was assumed based on previous studies on cells from skeletal muscle, that a local commitment of adult cells may result in more efficient cardiac differentiation than that previously observed with embryonic mesoangioblasts. Indeed, mouse cardiac mesoangioblasts show spontaneous (without chemical adjuvants) and high differentiation rate into beating cardiomyocytes while displaying only a low differentiation rate into smooth muscle cells. As for human cardiac mesoangioblasts, they show high differentiation into beating cardiomyocytes rate in the presence of 5-azatydine or when co-cultured with rat neonatal cardiomyocytes and only low differentiation rate into smooth muscle cells.
In the case of mouse cardiac mesoangioblasts, the efficiency of spontaneous cardiac differentiation is amazingly high and superior to already described cardiac stem cells (Anversa group, patent application WO 02/09650), Isl-1 positive cardioblasts (Chien group), Tert/Sca1+ progenitors (Schneider group, patent application WO 04/019767) and not even comparable to other types of stem cells whose cardiac differentiation ability is only anecdotic. Concerning their phenotype, mouse cardiac mesoangioblasts differ from all the other cardiac stem cells: a) they express CD34 and CD31 which is different from cardiac stem cells and Isl-2 cardioblasts; b) they express c-Kit and Nkx 2.5 which is different from Tert/Sca1 progenitors.
Human cardiac mesoangioblasts expressed similar markers and genes as mouse cardiac mesoangioblasts but these cells are only able to differentiate into cardiomyocytes in presence of 5-azatydine or in co-culture with rat neonatal cardiomyocytes.
Patents U.S. Pat. Nos. 5,486,359 and 6,184,035 describe human mesenchymal stem cells and methods for isolation and activation thereof, and control of differentiation from skeletal muscle stem or progenitor cells. The cells described in these patents are very different from the one of the present invention, in particular regarding the presence or absence of specific markers.